Khan et al A Figure I. Immunohistochemical analysis of Interaction of selected peptides with GP-2 nd internalization into PPs through M cells Anti-GP-2+EGFP-GbI Anti GP-2 EGFP-GbI was evaluated by immunohistochemical after administration of enhanced green fluorescent protein(EGFP) into ligated ops as described in the Materia ethods. (A)Whole-mounted PPs Anti-GP-2 +EGFP incubated with Gbl-EGFP and stained with DAPI op ligation and staining with GP-2 and FAE 4, 6-diamidino-2-Phenylindole(DAPI)as indicated Lower right panel shows merged image of anti-GP-2(red), EGFP-Gbl (green), and DAPl(blue).(C)Internalization EGFP-Gb1 EGFP- Gb2 f EGFPs assisted by selected ligands or without ligands as indicated, 60 min after gut loop assay: ()EGFP-Gbl,(EGFP-Gb2 (i)EGFP-Gb3, and (iM)EGFP alone. In each FAE case, the left and right panels show results rom light and fluorescence microscopy. ely, taken at lox magnification EGFP- Gb3 FAE, follicle-associated epithelium. Scale EGFPbars: 50 um stained whole PPs and frozen sections with anti-GP-2 after with the ligand-fused EGFPs increased the induction of incubation with either ligand-conjugated EGFPs or EGFP EGFP-specific serum IgG compared to EGFP alone. In par- alone. Immunohistochemical staining of the whole-mount ticular, Gb-1-conjugated EGFP enhanced the induction of and frozen sections of PPs confirmed the binding of only EGFP-specific IgG more efficiently and increased the level Gb-1-conjugated EGFP to M cells that specifically express of EGFP-specific serum gG by >2-fold compared to other iP-2(Fig. 1A, B). However, Gb-2 and Gb-3 conjugated to ligand-fused EGFPs(Fig 2A). The enhancement in immune EGFP were not preferentially bound to M cells. To confirm response was further analyzed at the IgG subclass level.As the ligand-assisted internalization of EGFPs into PPs, we shown in the Figure 2C, D, a-2-fold increase in the induc analyzed and compared the fluorescence intensity of EGFP tion of EGFP-specific IgGl was detected for Gb-l, but no fluorescence under the dome area of PPs in frozen sections significant increase in the level of Ag-specific IgG2a could I h after oral feeding. We found that ligand-fused EGFPs be detected even with the Gb-1-fused EGFP. were internalized into the PPs successfully; in particular, At the mucosal level. mice immunized with Gbl-fused peptide Gb-1 most efficiently transferred its fluorescence EGFP induced EGFP-specific fecal IgA level >1. 5-fold into PPs(Fig. IC). However, unconjugated EGFP did not stronger than those immunized with other ligand-fused show such internalization. These results show that Gb-1 EGFPs or with eGFP alone( Fig 2B).IL-4, IL-5, and IL-6 binds to GP-2 on M cells and can promote Ag transcytosis known to be associated with isotype switching to secretory IgA, oral immunization with Gb-1[AQ: 6] not only enhanced the induction of Gb-I-specific fecal IgA but also Gb-1 Enhances Ag-Specific Immune Response increased the number of Gb-1-specific IgA-secreting cells Induction in PPs compared with EGFP alone(Fig. 3B). In addition, oral administration of gbl-fused egfp also enhanced the To test the ability of the selected peptides to induce immune priming of EGFP-specific lymphocytes(Fig 3C response, we administered ligand-conjugated or only EGFF Interferon-Y(IFN-Y) is the representative cytokine for to mice orally and evaluated antibody response in systemic Thl-type response, and IL-4 and IL-6 represent the Th2 and mucosal compartments using ELISA. Immunization type response. We analyzed the pattern of cytokineKhan et al. 5 stained whole PPs and frozen sections with anti–GP-2 after incubation with either ligand-conjugated EGFPs or EGFP alone. Immunohistochemical staining of the whole-mount and frozen sections of PPs confirmed the binding of only Gb-1–conjugated EGFP to M cells that specifically express GP-2 (Fig. 1A,B). However, Gb-2 and Gb-3 conjugated to EGFP were not preferentially bound to M cells. To confirm the ligand-assisted internalization of EGFPs into PPs, we analyzed and compared the fluorescence intensity of EGFP fluorescence under the dome area of PPs in frozen sections 1 h after oral feeding. We found that ligand-fused EGFPs were internalized into the PPs successfully; in particular, peptide Gb-1 most efficiently transferred its fluorescence into PPs (Fig. 1C). However, unconjugated EGFP did not show such internalization. These results show that Gb-1 binds to GP-2 on M cells and can promote Ag transcytosis to PPs. Gb-1 Enhances Ag-Specific Immune Response Induction To test the ability of the selected peptides to induce immune response, we administered ligand-conjugated or only EGFP to mice orally and evaluated antibody response in systemic and mucosal compartments using ELISA. Immunization with the ligand-fused EGFPs increased the induction of EGFP-specific serum IgG compared to EGFP alone. In par￾ticular, Gb-1–conjugated EGFP enhanced the induction of EGFP-specific IgG more efficiently and increased the level of EGFP-specific serum IgG by >2-fold compared to other ligand-fused EGFPs (Fig. 2A). The enhancement in immune response was further analyzed at the IgG subclass level. As shown in the Figure 2C,D, a ~2-fold increase in the induc￾tion of EGFP-specific IgG1 was detected for Gb-1, but no significant increase in the level of Ag-specific IgG2a could be detected even with the Gb-1–fused EGFP. At the mucosal level, mice immunized with Gb1-fused EGFP induced EGFP-specific fecal IgA level >1.5-fold stronger than those immunized with other ligand-fused EGFPs or with EGFP alone (Fig. 2B). IL-4, IL-5, and IL-6 known to be associated with isotype switching to secretory IgA, oral immunization with Gb-1[AQ: 6] not only enhanced the induction of Gb-1–specific fecal IgA but also increased the number of Gb-1–specific IgA-secreting cells in PPs compared with EGFP alone (Fig. 3B). In addition, oral administration of Gb1-fused EGFP also enhanced the priming of EGFP-specific lymphocytes (Fig. 3C). Interferon-γ (IFN-γ) is the representative cytokine for Th1-type response, and IL-4 and IL-6 represent the Th2- type response.19,20 We analyzed the pattern of cytokine Figure 1. Immunohistochemical analysis of peptide–glycoprotein-2 (GP-2) interaction and internalization to Peyer’s patches (PPs). Interaction of selected peptides with GP-2 and internalization into PPs through M cells was evaluated by immunohistochemical staining of whole-mount specimens and cryosections of mouse PPs 20 min after administration of enhanced green fluorescent protein (EGFP) into ligated loops as described in the Materials and Methods. (A) Whole-mounted PPs incubated with Gb1-EGFP and stained with anti-GP2 antibody. (B) Frozen sections of mouse PPs prepared 20 min after gut loop ligation and staining with GP-2 and 4′,6-diamidino-2-phenylindole (DAPI) as indicated. Lower right panel shows merged image of anti–GP-2 (red), EGFP-Gb1 (green), and DAPI (blue). (C) Internalization of EGFPs assisted by selected ligands or without ligands as indicated, 60 min after gut loop assay: (i) EGFP-Gb1, (ii) EGFP-Gb2, (iii) EGFP-Gb3, and (iv) EGFP alone. In each case, the left and right panels show results from light and fluorescence microscopy, respectively, taken at 10× magnification. FAE, follicle-associated epithelium. Scale bars: 50 µm